The goal of this project is to test the feasibility of a new single-molecule DNA sequencing strategy that combines solid-state nanopores with mass spectrometry. The idea is to sequentially cleave each nucleotide or base from a DNA molecule as it transits a nanopore, then identify each one by determining its mass-to-charge ratio in a mass spectrometer. Identifying the bases of a translocating DNA molecule by mass spectrometry is appealing because: 1) It is an extremely sensitive technique that can easily distinguish the four DNA bases from their significantly different masses; 2) Modern ion detectors can detect the impact of single ions with a quantum efficiency approaching unity; 3) Those same ion detectors register ions as ~ 20 ns electrical pulses, offering a high detection bandwidth that may obviate any need to control the DNA translocation speed; 4) The sequence of DNA is revealed by the order in which ions of different mass impact the detector, and is not affected by variations of translocation speed; 5) Mass measurements are expected to be insensitive to the orientation of a base in the nanopore, which is difficult to control. The success of our strategy hinges on whether ionized bases or nucleotides can be controllably cleaved from the leading end of a DNA molecule as it translocates the nanopore, and transferred into a mass spectrometer that is housed in a vacuum chamber. This project consequently focuses on assembling a nanopore mass spectrometry instrument, and using it to understand and control ionization and molecular fragmentation processes at the liquid-vacuum interface. The specific aims are to: 1) Detect DNA mononucleotides in a quadrupole mass spectrometer coupled to a m-scale, chip-based pore; 2) Demonstrate mass spectrometry of single DNA bases ejected from a nanopore; 3) Identify efficient DNA fragmentation and ionization mechanisms; 4) Sequence short DNA homopolymers. Obtaining high quality sequence information (e.g. Q20 bases or better) from DNA homopolymers will demonstrate the viability of the nanopore mass spectrometry technique. This would justify developing a second-generation system, capable of sequencing long, heterogeneous DNA molecules. The $1000 per genome objective would be well within reach. Public Health Relevance: This project aims to develop a single-molecule DNA sequencing technology that would permit a full human genome to be sequenced for under $1000 and in under one day. This would have a profound impact on the life sciences by enabling genetic studies of exceptionally wide scope, and it would enable revolutionary health care options through personal genomics.